The conjugation of water-soluble polyalkylene oxides with therapeutic moieties such as proteins and polypeptides is known. See, for example, U.S. Pat. No. 4,179,337, the disclosure of which is hereby incorporated by reference. The '337 patent discloses that physiologically active polypeptides modified with PEG circulate for extended periods in vivo, and have reduced immunogenicity and antigenicity.
To conjugate polyalkylene oxides, the hydroxyl end-groups of the polymer must first be converted into reactive functional groups. This process is frequently referred to as “activation” and the product is called an “activated polyalkylene oxide.” Other polymers are similarly activated.
Commonly-assigned U.S. Pat. No. 5,730,990 describes one solution for addressing some of the problems associated with some polymer conjugation reactions. Specifically, it was determined that the polymer conjugate has a different pI than the unmodified protein, enzyme, etc. For example, PEGylation, i.e. attachment of the polymer to lysine epsilon amino groups results in a decrease in the isoelectric point and changes the pH optimum, i.e. the pH at which maximum bioactivity is observed. As reported in the '990 patent, it would be beneficial to restore the original pI or even alter the pI value of the polymer conjugate in order to optimize bioactivity at physiologic pH.
Over the years, it was determined that improvements in the synthetic procedure for preparing the activated polymers described in the '990 patent would be desirable, especially where a high degree of purity is required. Example 10 of the '990 patent indicates that reacting PEG-Cl and sarcosine at 750 for 4 days resulted in an N-methyl glycine PEG intermediate which was only 80% pure. It was also found that a portion of PEG-Cl reverted to PEG-OH, thus making it difficult to obtain high purity activated linkers or PEG conjugates made therewith.
In view of the foregoing, it would be desirable to provide improved methods for preparing activated polymers having alpha nitrogen groups. The present invention addresses this need.